Plasmid having temperature dependent plasmid copy number

ABSTRACT

Gene products of plasmid DNA, such as proteins, are prepared in high yields by cultivating bacteria carrying a plasmid which shows a controlled constant plasmid copy number at one temperature and a much higher or totally uncontrolled copy number at a different temperature. The plasmid may be prepared by recombinant DNA technique using a cloning vector showing the temperature dependent plasmid copy number pattern.

This is a division of application Ser. No. 908,108, filed May 22, 1978.

FIELD OF THE INVENTION

The present invention provides a process for producing a gene product ofplasmid DNA, to bacteria and plasmids useful in the process, to acloning vector which can be used in constructing recombinant DNAplasmids useful in the process, and to a method of preparing suchbacteria, plasmids, and cloning vectors.

BACKGROUND OF THE INVENTION

It is known to prepare useful polypeptides and proteins, for exampleenzymes, hormones, and (for use in e.g. vaccine preparation) toxins andother antigens, by cultivation of bacteria carrying plasmids with genescoding for the desired polypeptides or proteins. It is also known toconstruct plasmids containing desired genes by so-called recombinant DNAtechnique, which makes it possible to obtain, from the cultivatedbacteria carrying such recombinant DNA plasmids, gene products whichinherently are characteristic to other organisms than the bacteria usedas host cells. In the preparation of recombinant DNA, a so-calledcloning vector, that is, a plasmid which is able to replicate in thehost bacterium, is combined with a DNA fragment containing a gene orgenes coding for the desired product or products.

The recombinant DNA technique, in its most useful form, is based on thefollowing principle:

DNA can be cut into pieces in a very specific way by restrictionendonucleases. These pieces can then be joined to each other by DNAligase. DNA cloning utilizes a plasmid vector that is a circular DNAmolecule containing only one site for one or several restrictionendonucleases. Treatment of such a vector with a restriction enzymegives one species of a linear molecule. If this molecule is mixed with aDNA sample that is also treated with the same endonuclease, it ispossible by ligation to obtain molecules composed of the vector to whicha foreign DNA fragment has been fused. These plasmid molecules arecalled recombinant DNA. The vector with the foreign DNA can betransformed into a bacterial host cell, which means that it is taken upby and replicated in the bacterial host. Since the vector is able toreplicate, the foreign DNA is also replicated.

If the foreign DNA is transcribed and translated in the bacterial host,the gene products of the foreign DNA are produced in the bacterial host.This production is in general proportional to the gene concentrationwhich, on its side is proportional to the number of copies of therecombinant DNA plasmid molecules per cell. This means that in order toobtain large quantities of the desired gene products of the plasmid, ahigh number of copies of the plasmid per bacterial cell should be aimedat. It is known that some cloning vectors inherently replicate in a highcopy number per bacterial cell, up to about 100. However, if the geneproduct produced by the foreign DNA combined with such cloning vector isone which is not well tolerated by the bacterial host, there may bedifficulties in propagating a bacterial clone up to the desiredproduction size culture because of inhibition exerted by the geneproduct. On the other hand, even a copy number of the order of about20-100 will not always give rise to satisfactory yield of the desiredgene product in the production culture. It is known that the number ofcopies of plasmids can be further increased by amplifying the inhibitionof the protein synthesis, for example by addition of chloramphenicol,but as protein synthesis is necessary for preparing gene products of thecloned DNA, the amplified DNA will only be useful for formation of geneproducts thereof if the protein synthesis inhibiting component can beremoved again, which is not always possible and often a complicatedprocedure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a process for preparing a gene product ofa DNA plasmid, in which the plasmid is one which allows both effectiveplasmid amplification and obtainment of large quantities of the plasmidgene products. The invention utilizes plasmids having atemperature-dependent plasmid copy number pattern in that the plasmidshows a controlled constant plasmid copy number when host bacteriacarrying the plasmid are cultivated at one temperature, but whichplasmid shows, when the host bacteria carrying the plasmid are grown ata different temperature, an altered plasmid copy number pattern allowinga much higher or totally uncontrolled copy number. Hence, the number ofcopies of such plasmid is low at one temperature, which is an advantagesince it decreases the risk that the cloned plasmid or its gene productsshould disturb growth of the host bacterium. However, the amount of theplasmid can be rapidly increased by a simple temperature shift, wherebysimultaneous formation of the cloned plasmid and its gene products isobtained, and the production of gene products of the plasmid proceedsrapidly.

In accordance with this, the invention provides a process for producinga gene product of plasmid DNA, said process comprising cultivatingbacteria carrying a plasmid showing a controlled constant plasmid copynumber when the host bacteria are cultivated at one temperature, andshowing, when the host bacteria carrying the plasmid are grown at adifferent temperature, an altered plasmid copy number control allowing amuch higher or totally uncontrolled copy number; under conditionsincluding at least a period of cultivation at or approaching atemperature at which the plasmid shows an altered copy number controlallowing a much higher or totally uncontrolled copy number; andharvesting, from the bacterial culture, a gene product of the plasmid.

The crux of the present invention is the utilization of the particulartype of plasmid having the temperature-conditioned plasmid copy numberpattern and the recognition that this type of plasmid, when copied in avery high number at one temperature, also gives rise to production oflarge amounts of its gene products. The cultivation per se is suitablyperformed using conventional techniques, including conventional nutrientmedia which are known to be optimal to the bacterial species inquestion, and also, the harvesting of the gene products is performed inaccordance with well-known methods adapted to the identity andproperties of the particular gene product prepared, the properties ofthe host bacteria, etc. Special and critical to the process of thepresent invention is the temperature regulation involving at least aperiod of cultivation at or approaching a temperature at which theplasmid shows an altered copy number pattern allowing a much higher ortotally uncontrolled copy number, in other words, involving a period ofcultivation during which the plasmid is copied in a high number ofcopies, and, as it has been found, during which gene products of theplasmid are formed in correspondingly high amounts.

The plasmid having temperature-dependent plasmid copy number pattern maybe one prepared by recombinant DNA technique using a cloning vectorshowing the temperature-dependent copy number pattern, or the plasmidmay be one obtained by mutagenization of an existing plasmid havinggenes for a desired production.

A detailed description of plasmids showing the above-mentionedtemperature-dependent plasmid copy number pattern has not been givenpreviously and appears from the below examples which describe thepreparation of some plasmids of this kind, including plasmids useful ascloning vectors.

The plasmid showing the above-mentioned temperature-dependent plasmidcopy number pattern with controlled constant copy number per cell at onetemperature and a much higher or totally uncontrolled copy number (inthe following often termed "runaway-replication") at another temperaturemay be prepared by mutagenization of an existing plasmid which is knownto replicate autonomously in the host bacterium in question. Inaccordance with known principles, the mutagenic treatment can beperformed in vivo or in vitro, and while the mutagenic agents so farused appear from the examples, it is assumed that the kind of mutagenictreatment is not critical. After the mutagenic treatment (and, if themutagenic treatment was performed in vitro, transformation of themutagenized material into the host bacterium), a bacterial clone inwhich the plasmid copy number is controlled when the bacteria arecultivated at one temperature and in which the plasmid copy number ismuch higher or totally uncontrolled when the bacteria are grown underdifferent temperature is then to be isolated. The isolation proceduremay be based upon screening at two temperatures between which a shift inplasmid number copy control pattern is sought for. One indication usefulin such screening is the fact that on a substrate which is known to benon-limiting to the host bacterium in question, growth inhibitionespecially at a particular temperature is likely to be due to theproduction of a large number of copies of plasmid and/or of largeamounts of gene products thereof.

It has been found that a more suitable way of isolating a plasmid mutantshowing runaway-replication pattern is often to use a two-stagemutagenic treatment comprising a first stage in which a plasmid-carryingbacterial clone is isolated in which the plasmid shows one plasmid copynumber control pattern at one temperature and another plasmid copynumber control pattern permitting a higher copy number at a differenttemperature, and a second stage to obtain, from the mutant obtained fromthe first stage, a plasmid-carrying bacterial clone in which the plasmidshows a much higher or totally uncontrolled copy number at said second(different) temperature. This procedure allows optimum utilization ofsuitable selection techniques, for example double antibiotic selection:

One type of plasmid copy mutants has the following characteristics: Atlow temperature (e.g. 30° C.), the copy number is close to that of theparent plasmid, while at higher temperature (e.g. 40° C.), the copynumber is about 4-fold higher. A suitable isolation procedure used toisolate such mutants makes use of the fact that the resistance towardsampicillin and chloramphenicol is directly proportional to theconcentration of genes coding for the corresponding resistance enzymes(Uhlin et Nordstr/o/ m, Plasmid 1, 1977), and of the fact thatampicillin kills only bacteria which are in a phase of growing.

It has been found that strains containing plasmid R1drd-19 (which isdescribed in greater detail below) exhibit a single cell resistance onLA plates of about 100 μg ampicillin/ml and of about 100 μgchloramphenicol/ml. The resistance in liquid medium towards these twoantibiotics is about the same as on LA plates. The resistance towardsampicillin and chloramphenicol is directly proportional to the geneconcentration (Uhlin et Nordstr/o/ m, Plasmid 1, 1977). This effect hasbeen used to isolate mutants of plasmid R1drd-19 with an increased copynumber, so-called copy mutants. However, the combination of abacteriostatic and a bacteriocide can also be used to isolate copymutants with different copy numbers at different temperatures, as thisselection method allows easy counter-selection of both parent plasmidand non-conditional copy mutants. The following procedure was found tobe a suitable one: It is assumed that a copy mutant exists with a lowcopy number at a low temperature (30° C.) and a high copy number at ahigh temperature (40° C.) (the procedure is, however, not limited tothis type of copy mutant, but can also be used to isolate other possibletypes of temperature-dependent copy mutants).

The culture of the plasmid-containing cells is grown at 30° C., andchloramphenicol is added to a concentration (300 μg/ml) which inhibitsthe growth of cells containing plasmids with normal copy number. Thecells containing plasmids with a high copy number at 30° C. are killedwith ampicillin (4000 μg/ml). The surviving cells are collected and thetemperature is raised to 40° C. Cells containing plasmids with a highcopy number at 40° C. are selected on ampicillin plates (500-2000μg/ml). Among the cells surviving, some should containtemperature-dependent plasmid copy mutants.

Cells selected in the above manner can then be subjected to furthermutagenic treatment, and a suitable method for selecting, among the thusmutagenized cells, a mutant having runaway behaviour at the secondtemperature has been found to comprise an initial selection for cellswhich, at the lower temperature, show a controlled and constant, butincreased copy number, indicating that the replication control systemhas again been subject to mutation.

The particular type of mutation which has occurred in the specificplasmids illustrated in the examples is not yet known with certainty,but it is believed that an effect of the mutation is that a proteinmediated by the plasmid and involved in the plasmid copy number controlhas, due to the mutation, become modified into a form which is notstable at the higher temperature. While it is evident that the mostpractical embodiment of the plasmid of the invention, when it is to beused as a cloning vector, is one which, like the cloning vectorsdescribed in the examples, in itself contains all elements necessary forthe temperature-dependent copy number behaviour, it is also obvious thatthe principles of the present invention and advantages conferred therebymay also manifest themselves when the mutation is of the nonsense typecombined with corresponding temperature-dependent nonsense suppressor inthe host bacterium, for example when the plasmid is an amber mutant andthe host bacterium is one which shows temperature-dependent ambersuppressor effect.

The temperature at which the plasmid shows runaway behaviour will notnecessarily be one which is higher than the temperature at which theplasmid shows a controlled constant copy number, also the reversesituation is possible. If it is desired to prepare a mutant showingrunaway replication at a lower temperature than the temperature givingcontrolled constant copy number, the selection or screening criteria areadapted correspondingly, However, when the gene product to be preparedby cultivating the plasmid-containing bacteria is one which is notdeteriorated at the higher temperature, it is preferred that thetemperature at which the plasmid shows runaway replication is a highertemperature than the one giving a controlled constant plasmid copynumber. When this applies, the amplification of the plasmid takes placeunder the same conditions allowing the relatively highest cell growthrate. The plasmids illustrated in the working examples were designed toshow, in the mesophilic bacteria exemplified by Escherichia coli, aconstant controlled plasmid copy number at 30° C., and runaway behaviourat 40° C. As will appear from the data on these plasmids, the constantcontrolled plasmid copy number is retained up to about 32° C. for theseplasmids and the temperature at which the plasmid shows runawaybehaviour is above about 36° C.

The most interesting plasmids of the present invention are plasmidswhich show a reasonably large, but constant copy number at onetemperature, and a copy number which is at least 20 times higher at thedifferent temperature. Whether or not total runaway behaviour withresulting death of the cell is obtained will to some extent depend uponthe bacterial host and its ability to tolerate the plasmid and its geneproducts in high concentration.

Hence, the present invention permits, on the one hand, obtainment ofplasmid copy numbers which were hitherto unobtainable, up to the orderof several thousand per cell, and, as it has been found, concomitanthigh production of gene products of the plasmid during a sufficientnumber of generations (usually 4-6) of growth at the high copy number tosecure a considerable production of gene products. On the other hand,the invention provides a most simple control of the copy number, whichmay be utilized in various manners, depending upon the individualconditions with respect to bacterial host, desired gene product, etc.:In most cases, it will be preferred that the propagation of the bacteriafrom an individuum or a clone up to a production size culture isperformed at or near the temperature at which the plasmid shows acontrolled constant copy number, in order to avoid any inhibition of thebacterial growth by an increasing plasmid and gene productconcentration. Thereafter, the temperature may be shifted to atemperature at which the plasmid shows an altered copy number controlallowing a much higher or totally uncontrolled copy number, and after asuitable production period, often until the growth of the bacteria isinhibited by the production of the plasmid and/or gene products thereof,the harvesting of the gene product is performed. Depending on theparticular conditions, it may be desired to perform the productioncultivation at a temperature which is only approaching the temperatureat which the plasmid shows the altered copy number control, so as toobtain a steady-state culture giving a high yield of gene product (whichmay then be continuously or intermittently harvested from the culture ina manner known per se), but at which temperature the host cells surviveand are still capable of propagation.

One example of the unique control possibilities obtained through thepresent invention is preparation of a temperature-sensitive protein asthe desired gene product. In such case, the bacterium may be propagatedup to production size culture at the temperature at which the plasmidcopy number is controlled and constant, and thereafter, by a temperatureshift to the runaway temperature, the plasmids may be amplified.Subsequent to amplification of the plasmids and while the cells arestill viable and capable of propagation, the temperature is againshifted to the lower temperature giving constant copy number, and atthis lower temperature, the resulting bacteria carrying the much highernumber of plasmids per cell are used for the production of thetemperature-sensitive protein in question. It is known that there aremany organisms which are not viable at temperatures above about 30° C.,and the proteins of which, therefore, may be denatured at highertemperatures. Therefore, this embodiment of the temperature regulationmay prove especially important when gene products inherent to suchorganisms are to be prepared by recombinant DNA/cloning technique.

As indicated above, the plasmid showing the characteristictemperature-dependent copy number behaviour involving runawayreplication may be one derived by mutagenic treatment, from a parentplasmid possessing genes for the desired production, but without thecharacteristic runaway behaviour. However, for many practical purposesthe plasmid used in the production cultivation will be one prepared byrecombinant DNA technique, using as a cloning vector a suitable plasmidshowing the characteristic temperature-dependent runaway replication.This combines the advantages of the conventional recombinant DNAtechnique with the advantages of the temperature control and the veryhigh obtainable number of plasmid copies per cell, with the consequentamplified production of polypeptide or protein mediated by the foreignDNA fragment. It is contemplated that the very high number of plasmidcopies obtainable by using a cloning vector of the present inventionwill permit production of large or at any rate reasonable amounts ofproteins which, due to their being genetically relatively remote fromthe host bacterium in question, could hitherto not be produced at all,or at any rate not be produced in a satisfactory amount, by cloningtechniques. Hence, the cloning vector aspect of the present invention isa very important aspect.

In order to be useful as a cloning vector, the plasmid should show, forat least one restriction endonuclease, one and only one site susceptibleto the endonuclease, and the said site should be one which afterinsertion of a fragment of foreign DNA at this site, permits theresulting recombinant DNA to replicate autonomously and, to obtain theadvantages of the present invention, with retention of the capability ofshowing the temperature-dependent copy number pattern.

The most suitable restriction endonucleases for use in recombinant DNAtechnique are those giving the so-called "cohesive ends" on both thecloning vector and the DNA fragment, in other words, single strandedregions at the ends of the molecules with complementary base sequenceallowing base pairing to identical sequences.

As appears from the examples, cloning vectors having one site forrestriction endonuclease have been prepared, and also vectors having onesite for one restriction endonuclease, and another site for anotherrestriction endonuclease, has been prepared. An advantageous way ofconstructing cloning vectors fulfilling the above conditions is often toprepare, from a larger plasmid showing the temperature-dependent runawayreplication, a "miniplasmid" of sufficiently small size to show only onesite susceptible to a useful restriction endonuclease, but withsufficient size to still contain the genes indispensable to the specialtemperature-dependent replication behaviour. Such miniplasmids may beprepared from a larger plasmid by isolating bacterial clones carryingeither spontaneously occurring or in vitro-prepared (and thereaftertransformed) miniplasmid derivatives of the parent plasmid. Theisolation of the desired miniplasmids (or bacteria containing them) isperformed by suitable and well-known screening and/or selection methods.

In principle, an ideal cloning vector is one which contains as few genesas possible coding for the production of non-desired proteins. However,for many purposes, it is desired that the cloning vector contains genesmediating a so-called marker useful for identification and/or selectionof cells carrying the plasmid. The most useful marker is antibioticresistance, for example ampicillin resistance, as this permits, after atreatment for transforming a recombinant DNA into a bacterial host, aneasy counter-selection of bacteria which have not received therecombinant plasmid. However, also cloning vectors without a marker maybe useful cloning vectors, for example when the genes to be inserted bythe recombination in themselves carry a marker.

When it is desired to introduce a marker, for example antibioticresistance, in a plasmid of the present invention to be used as acloning vector, this may be done by transposition of a DNA fragment in amanner known per se. An example of such transposition is illustrated inExample 5. Another way of introducing a foreign DNA fragment mediating amarker function would be by recombinant DNA technique, but this wouldconsume a restriction site on the cloning vector and can, therefore,only be used in the cases where the cloning vector has at least tworestriction sites useful in recombinant DNA technique.

Another embodiment of the preparation of a cloning vector of the presentinvention is to introduce, by mutagenization, the above-describedtemperature-dependent runaway replication in an existing cloning vectorknown to function well in a desired organism, for example the plasmidpSC101 which is a well-established cloning vector in E. coli.

METHODS USED IN THE EXAMPLES

Several Escherichia coli K-12 strains (Table 1) and plasmids (Table 2)were used.

The experimental techniques used were standard techniques used inmicrobial genetics (J. Miller, Methods in Molecular Biology, Cold SpringHarbor Laboratory) and in genetic engineering (T. Tanaka and B.Weissblum, J. Bacteriol. 212 (1975) 354-362).

                                      TABLE 1    __________________________________________________________________________    Escherichia coli K-12 strains    Strain         Genotype       Reference or Source    __________________________________________________________________________    EC1005         thi, met, nal, relA                        Grindsted et al, J. Bact. 110 (1972) 529.    D11  thi, his, pro, trp, strA                        Boman et al, Genet. Res. 12 (1968) 169.    C600 thi, lac, leu, thr                        Appleyard, R. K., Genetics 39 (1954) 440.    UB1731         thi, met, pro, amp (Tn802), nal                        Bennett, P. M. and M. H. Richmond, J. Bact. 126                        (1976) 1.    1100 thi, endoI    __________________________________________________________________________

                  TABLE 2    ______________________________________    Plasmids used    Plasmid   Source    ______________________________________     ##STR1## Meynell * Datta, Nature 214 (1967) 885.    pSF2124   So et al, & gen. Genet. 142 (1975) 239.    ______________________________________

Plasmid R1 is a transferable plasmid that mediates resistance to thefollowing antibiotics: ampicillin, chloramphenicol, kanamycin,streptomycin, and sulphonamides. The plasmid has a molecular weight of65×10⁶ daltons. Normally, in Escherichia coli, the plasmid is present inabout one copy per chromosome equivalent. It is possible by mutation inthis plasmid to increase the copy number several fold above this level,so-called copy mutants (K. Nordstr/o/ m et al., J. Bacteriol. 110,562-569 (1972)).

In the examples, the following stages are described:

Example 1: Isolation of a plasmid mutant (pKN301) which has atemperature-dependent replication control. The copy number of thisplasmid increases about 4 fold at higher temperature.

Example 2: The isolation from plasmid pKN301 of a mutant (pKN400) thatlacks replication control at higher temperature (a runaway mutant).

Example 3: The isolation from plasmid pKN400 of a miniplasmid (pKN402)that retains the runaway behaviour but carries only one site for therestriction endonuclease EcoR1.

Example 4: The isolation from a derivative of plasmid pKN400 of anotherminiplasmid that retains the runaway behaviour but carries only one sitefor the restriction endonuclease EcoR1.

Example 5: The insertion, by translocation, of an antibiotic resistancemarker into miniplasmid pKN402 to obtain miniplasmid pKN403.

Example 6: Insertion of a Streptomycin resistance gene on miniplasmidpKN403 and cloning.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the properties of a runaway-replication plasmid mutant.Strain D11 carrying plasmid R1drd-19 (o) or pKN400 (Δ) was grownlogarithmically in LB medium at different temperatures.

FIG. 1a The amount of covalently closed circular (ccc) DNA was analysedby ethidium bromide cesium chloride gradient centrifugation afterlabelling with ³ H-thymidine. The plasmid DNA content was calculated as% of chromosomal DNA.

FIG. 1b The total amount of DNA and protein of the cells was measuredchemically and the ratio DNA/protein is shown in a relative scale withthe value for strain D11-R1drd-19 at 30° C. set for 100%.

FIG. 1c Growth rate (doublings/hour) was determined by measuring theoptical density in a Klett-Summerson colorimeter.

FIG. 2 shows the relative increase in plasmid mediated β-lactamasecontent and total protein of strain D11-pKN400 at 40° C. A culturegrowing logarithmically in LB-medium at 30° C. was shifted to 40° C.During the subsequent growth, samples were taken and assayed for amountof β-lactamase and total protein. The relative increase after thetemperature shift is plotted on a logarithmic scale.

FIG. 3 shows the plasmid DNA content of strain C600-pKN402. Cultures ofC600-pKN402, growing in LB-medium and labelled with ³ H-thymidin, wereanalysed by cesium chloride-ethidium bromide centrifugation.

FIG. 3a shows the result after logarithmic growth at 30° C. and

FIG. 3b shows the result after 3 hours of growth at 40° C.

The density of the gradients is increasing from right to left and thetotal number of fractions was 63 (FIG. 3a) and 52 (FIG. 3b),respectively.

FIG. 4 shows EcoR1 restriction fragment analysis of in vitro constructedhybrid plasmids. Purified plasmid DNA was digested with EcoR1endonuclease and the fragment pattern was analyzed byagarose-gel-electrophoresis. After staining in ethidium bromidesolution, the gel was photographed under illumination of UV-light. Fromleft the plasmids are: the cloning vehicle pKN403, two pKN403 basedhybrid plasmids (pKN404, pKN405), the plasmid R1drd-19 as reference andplasmid pKN402 for comparison. The denotions of the EcoR1 fragments ofR1drd-19 are shown to the right.

EXAMPLE 1

A 40 ml culture of strain EC1005 containing the plasmid R1drd-19 wasgrown at 30° C. in casamino acids medium. At a cell density of about 10⁸cells/ml the cells were chilled and harvested by centrifugation at 4° C.The cells were washed once in isotonic NaCl solution, resuspended in 1Mhydroxylaminehydrochloride (pH 6.0), and incubated at 37° C. for 20 min.After the mutagenization the culture was cleared of the mutagen anddiluted in LB medium (30° C.) to a cell density of about 10⁷ cells/ml.This culture was divided into 4 aliquots and incubation was continued at30° C. After 60 min. at 30° C. chloramphenicol (300 μg/ml) was added and10 min. later ampicillin (4000 μg/ml). Incubation was continued foranother 60 min. The surviving cells were harvested, washed twice with LBmedium (40° C.) and resuspended in LB medium (40° C.) to a cell densityof about 5×10⁸ cells/ml. The cultures were incubated for 30 min. at 40°C. and the cells in the cultures were then plated on LA platescontaining different concentrations of ampicillin (300-2000 μg/ml). Theplates were incubated over night at 40° C. and the surviving coloniesisolated and tested for their resistance pattern at 30° C. and 40° C.Cells possessing an ampicillin resistance of at least 500 μg/ml at 40°C. were found with a frequency of 10⁻⁶. Four hundred colonies isolatedfrom 500-2000 μg ampicillin/ml were picked, subcultured and tested fortemperature-dependent resistance. Five clones, 4 of them from individualsubcultures, showed more than a 3-fold difference in resistance between30° C. and 40° C. After transfer of the plasmids to an unmutagenizedstrain EC1005, resistance to ampicillin, streptomycin andchloramphenicol was determined. All 5 clones showed a 3-5-fold increasedresistance to all three antibiotics at 40° C. compared to 30° C. (Theparent plasmid exhibited resistances of 75 and 125 μg ampicillin/ml at30° C. and 40° C., respectively). One of the mutants, pKN301, wasanalyzed in greater detail. This plasmid has the same molecular weightas the parent plasmid, R1drd-19 (65×10⁶ daltons).

Copy number analysis of plasmid pKN301

Plasmid content was measured in two ways, by determination of CCC-DNAand of specific activity of β-lactamase. The amount of CCC-DNA for eachplasmid under different growth conditions was measured both as theamount of fast-sedimenting material in an alkaline sucrose gradient andas the amount of satellite band in a dye-CsCl buoyant density gradient(Table 3). The mutant, when grown at high temperature, showed 3 to 4times as much plasmid DNA as the same bacterium carrying plasmidR1drd-19, while at low temperature the amount of plasmid DNA was closeto that of the parent plasmid.

                  TABLE 3    ______________________________________    Plasmid copy number of cells carrying plasmid    pKN301 at different temperatures.sup.(a)    Temperature   Copy number.sup.(b)    (°C.)  pKN301    ______________________________________    30            1.2    34            1.3    35            1.7    37            4.0    39            4.1    40            3.4    44            3.7    ______________________________________     .sup.(a) Strain EC10005 was used as host.     .sup.(b) Copy number was ascertained by determining the CCCDNA/chromosoma     DNA ratio on dye/CsCl density gradients.     ##STR2##             which is set to 1.0 at each temperature.

EXAMPLE 2 Isolation of a runaway-replication plasmid mutant (pKN400)

As described in Example 1, it is quite evident that when the mutation inplasmid pKN301 is expressed, the copy number is still carefullycontrolled. This temperature-dependent copy number mutant has been usedas the parent plasmid in the search for plasmid mutants which have nocontrol of the copy number at a condition where the mutation isexpressed. A second mutation that affected the copy number level ofpKN301 at 30° C. was introduced and then tests for the behaviour of themutant at higher temperatures were carried out:

Strain 1005 carrying plasmid pKN301 was mutagenized with ethyl methanesulphonate and clones with increased resistance to ampicillin wereselected on plates at 30° C. Then the clones were tested at differenttemperatures and some of the isolates were found to have a drasticallyreduced viability at 37° C. and 42° C. By transfer of the plasmid to anunmutagenized host strain by conjugation it was shown that the reducedviability was caused by the mutant plasmid. One of the isolates waschosen for further work. In the transfer to the unmutagenized host(strain D11) selection for the plasmid recipient was done by selectingcells resistant to chloramphenicol (25 μg/ml), and the recipient nowcarrying the plasmid was subsequently tested for the presence of otherplasmid-mediated resistances. The plasmid of a clone that did show allR1-mediated resistances was denoted pKN400. Some of the properties ofpKN400 in strain D11 are illustrated in FIG. 1, which summarizes theresults after the cells were grown at different temperatures. There wasa drastic increase in the amount of plasmid DNA at temperatures above34° C. (FIG. 1a). A similar increase was shown for the total amount ofDNA in the cells (FIG. 1b), and there was a corresponding decrease inthe growth rate of the cells (FIG. 1c). The bacteria did not grow at ameasurable rate at temperatures above 35° C.

By shifting a culture growing at 30° C. to 37° C. (or 40° C.) it hasbeen shown that at the higher temperature there is no copy numbercontrol left to regulate the plasmid replication. The plasmid copynumber then increases logarithmically which eventually becomes lethalfor the host cell. An important finding is that, during such atemperature shift, the plasmid genes are expressed throughout the entireperiod when the amount of plasmid increases. The resulting gene dosageeffect is illustrated in FIG. 2, which shows the increase in totalamount of protein and of the plasmid mediated β-lactamase.

EXAMPLE 3 A miniplasmid with temperature-dependent runaway-replication(pKN402)

Some years ago it was found that a plasmid copy mutant couldspontaneously give rise to smaller plasmid segregants, miniplasmids,that were able to replicate automonously (Goebel and Bonewald, J. Bact.123 (1975), 658-665. Later, a large number of such miniplasmids havebeen isolated from various copy mutants. Various sizes of miniplasmidshave been obtained with molecular weights down to about 4×10⁶ daltons.The runaway-replication mutant plasmid pKN400 was found to be a goodsource for the isolation of miniplasmids, especially if uncontrolledplasmid replication was allowed by growth of the host cells at hightemperature.

After a transfer of pKN400 to strain C600 some clones appeared to bevery unstable with respect to plasmid maintenance as judged by tests (at30° C.) for plasmid mediated antibiotic resistance. However, in somecases the temperature sensitive phenotype was retained although thecells no longer carried resistance to the antibiotics. Byagarose-gel-electrophoresis it was shown that these cells did carry aplasmid, and in all cases the molecular weight was about 5×10⁶ daltons.One such clone was chosen for further work and the plasmid was denotedpKN402. Temperature shift experiments showed that this miniplasmidexpressed the same temperature-dependent behaviour as the large plasmid(pKN400). The amount of plasmid DNA in strain C600/pKN402 was analyzedby cesium chloride-ethidium bromide density gradient centrifugationafter growth at 30° C. (FIG. 3a) and after about 3 hours of growth at40° C. (FIG. 3b). Since the molecular weight of pKN402 was estimated tobe 4.65×10⁶ daltons it can be calculated that there are about 50 plasmidcopies per cell at 30° C. and after the temperature shift the numberincreases to about 5000 copies per cell. For analysis with restrictionendonucleases the DNA of pKN402 was purified by Ethidium Bromide/CesiumChloride gradient centrifugation. It was found that digestion with theEcoR1 endonuclease yields one linear molecule; i.e. there is one sitesusceptible to the enzyme on pKN402 and that is a very important factfor the considerations of the usefulness of this plasmid as a cloningvehicle.

The strain E. coli K12 C600/pKN402 is deposited in the GermanMicroorganism Collection (Deutsche Sammlung von Mikroorganismen,Grisebachstr. 8, D-3400 G/o/ ttingen), hereinafter called DSM, underaccession No. 1228.

EXAMPLE 4 Another miniplasmid with temperature-dependent runawayreplication (pKN410)

Another miniplasmid derived from pKN401 (a derivative of pKN400, whichhas lost kanamycin-resistance) was isolated by an alternative method.Plasmid DNA (pKN401) was digested in vitro with the restriction enzymeEcoR1 and thereafter treated with the enzyme DNA ligase in order to fuseand seal the ends of the generated linear DNA fragments. The DNA wastransformed to E. coli C600 cells and clones resistant to ampicillinwere isolated and tester further. One of these clones having a phenotypesimilar to E. coli cells carrying the plasmid pKN401 (temperaturesensitivity) was shown to carry a plasmid of the size 12×10⁶ daltons(pKN410).

The copy number of this plasmid was found to be about 20 copies per cellat 30° C. and about 2000 copies per cell at temperatures above 37° C.

The presence of the ampicillin resistance gene on the plasmid wasverified by transformation. Restriction enzyme analysis showed thatpKN410 carried only one site susceptible to the restriction enzymeEcoR1, and only one site susceptible to the restriction enzyme BamH1.

Cultures of cells carrying pKN410 showed exactly the same growth patternafter a temperature shift from 30° C. to 40° C. as the parent plasmidpKN400, and there was a similar increase in the amount of theplasmid-mediated β-lactamase.

The strain E. coli K12 C600/pKN410 is deposited in the GermanMicroorganism Collection (DSM) under accession No. 1230.

EXAMPLE 5 Introduction of an ampicillin resistance gene in miniplasmidpKN402 to obtain miniplasmid pKN403

The plasmid pKN402, though having the important properties oftemperature dependent replication and the presence of a single site forthe restriction enzyme EcoR1, does not possess a marker which can beused when selecting for bacterial clones harboring the plasmid. Sincethe step next to the in vitro joining of DNA fragments with the vectoris a transformation of DNA to competent bacterial cells it is anadvantage that transformed cells can be easily found. Therefore mostcloning vectors carry an antibiotic resistance gene which enables cellshaving the vector to survive in a medium containing the correspondingantibiotic. A plasmid from pKN402 which carries the gene coding forβ-lactamase, i.e. the cloning vector is resistant to penicillins, wasprepared:

The first step in the construction of the penicillin resistantderivative of pKN402 was to bring the TnA transposon to the E. Colistrain carrying pKN402. (For a description of transposons, consultKleckner, Cell 11 (1977) 11-23).

This step was performed as a transduction with the bacteriophage P1. Thestrain E. coli K12, UB1731 carries the transposon Tn802 (TnA) on thebacterial chromosome.

A culture of this strain was infected with the bacteriophage P1 and aphage stock prepared. This P1-stock was used to transduce the strainC600/pKN402. The cells infected with P1 were spread on plates containingampicillin (50 μg/ml). Colonies growing on these plates were purifiedand tested for ampicillin resistance and temperature sensitivity. (Allmethods are described in J. Miller: Methods in Molecular Biology). WhenTn802 was inserted in the chromosome of E. coli, the cells becameresistant to ca. 300 μg/ml benzylpenicillin. However, when thetransposon is inserted in a multi copy plasmid the level of resistanceis much higher. Therefore, there was selected for clones among thetransduced C600/pKN402 that were resistant to 1000 μg/mlbenzylpenicillin. Since pKN402 at 30° C. is present in ca. 50 copies percell, it was expected that translocation of Tn802 to pKN402 would resultin a higher level of penicillin resistance. 4 colonies of C600/pKN402growing on 1000 μg/ml benzylpenicillin were selected for furtheranalysis. When analysing the plasmid content of these 4 clones one hadonly one type of plasmid with a molecular weight of ca. 8×10⁶.

(The insertion of Tn802 results in an increase of the molecular weightof the plasmid of 3.2×10⁶). This clone was picked for further analysisand the plasmid termed pKN403. In order to verify that pKN403 had allthe properties of pKN402 and in addition carried the Tn802 transposon,plasmid DNA was prepared from the strain C600/pKN403, and this DNA wastransformed to another strain of E. coli K12. The transformed cells weregrown on plates containing ampicillin (50 μg/ml), and a colony growingon such a plate was analyzed for the presence of the plasmid and fortemperature sensitivity. It was found that the ampicillin resistantcells contained a plasmid of the same size as pKN403 and they weretemperature sensitive. The DNA of pKN403 was analyzed with respect tothe number of sites for the restriction enzymes EcoR1 and BamH1 and itwas found that like pKN402 the plasmid pKN403 had only one EcoR1 site.In addition pKN 403 carries one cut site for BamH1 (presumably in theTn802 sequence). Thus, at least two enzymes may be used when applyingpKN403 as a cloning vector.

In shifts from 30° C. to 40° C. the culture stops growing soon after.The copy number may be kept at a high steady-state level by using anintermediate temperature (e.g. 34° C.).

The strain E. coli K12 C600/pKN403 is deposited in the GermanMicroorganism Collection (DSM) under accession No. 1229.

Summary of the properties of the temperature-dependent cloning vectors

The examples describe the isolation and characterization of 3 smallplasmids which all may be used as cloning vectors.

One plasmid (pKN402) lacks any antibiotic resistance marker and maytherefore be used when the DNA to be inserted in the vector has agenetic marker useful in selection, or when screening for plasmids withincreased molecular weight is performed subsequent to the recombination.This vector may be very useful in the construction of other vectors, orin recombination with DNA carrying different genetic markers. In theplasmid pKN403 the presence of an ampicillin resistance gene makes aselection for plasmid carrying cells easy. Finally, the plasmid pKN410,carrying the ampicillin resistance gene, shows the growthcharacteristics of pKN400 after a temperature shift to 40° C. Thisplasmid is present in a low number per cell (20 copies/cell) at 30° C.

All of the plasmids have one site susceptible to the restriction enzymeEcoR1.

EXAMPLE 6 Cloning of a streptomycin resistance gene on plasmid pKN403

A cloning experiment was performed with the vector pKN403 in which anEcoR1 fragment carrying the gene mediating streptomycin resistance wasinserted into the vector. A previously constructed hybrid plasmidconsisting of the plasmid pSF2124 and the EcoR1 fragment G from plasmidR1drd-19 was used as the source of the streptomycin resistance gene.Purified DNA of the vector pKN403 and the mentioned hybrid plasmid wasmixed and digested with the EcoR1 endonuclease in order to obtain linearfragments. The DNA was then treated with polynucleotide ligase and usedin transformation of E. coli cells. Selection was made for cells havingresistance to ampicillin and streptomycin at 30° C. Clones so obtainedwere tested for growth at 40° C. and most of them were found to beunable to grow at this temperature. Further analysis of such clonesshowed that they carry plasmids of exactly the molecular weightcalculated for a hybrid plasmid consisting of plasmid pKN403 and thefragment G. FIG. 4 shows the EcoR1 fragment pattern of two such hybridplasmids (pKN404, pKN405). It was also shown that the amplification ofplasmid DNA at 37°-40° C. in case of pKN404 was similar to that foundfor pKN403.

It was therefore concluded that the insertion of a DNA fragment into theEcoR1 site of the vector thus constructing recombinant DNA molecules,does not alter the advantageous properties of the vector concerningamplification of DNA and/or gene products.

We claim:
 1. A recombinant DNA plasmid showing a controlled constantplasmid copy number when host bacteria carrying the plasmid arecultivated at one temperature, and showing, when the host bacteriacarrying the plasmid are grown at a different temperature, an alteredplasmid copy number control allowing an at least 20 times higher ortotally uncontrolled copy number.
 2. A plasmid as claimed in claim 1which shows an altered copy number control allowing an at least 20 timeshigher or totally uncontrolled copy number at a higher temperature thanthe temperature at which the plasmid shows a controlled constant plasmidcopy number.
 3. A plasmid as claimed in claim 2 showing a constantcontrolled plasmid copy number at a temperature up to about 32° C. andshowing an altered copy number control allowing an at least 20 timeshigher or totally uncontrolled copy number at temperatures of about 36°C.
 4. A plasmid as claimed in claim 1 and carrying a marker useful foridentification and selection of cells carrying the plasmid.
 5. A plasmidas claimed in claim 4 wherein the marker is a gene that mediatesantibiotic resistance to the host bacterium.
 6. A plasmid as claimed inclaim 5 in which the antibiotic resistance is ampicillin resistance. 7.A plasmid as claimed in any of claims 1-3 and 4-6, wherein the plasmidis a mini-plasmid.
 8. A plasmid as claimed in claim 1, wherein theplasmid is a derivative of R1.
 9. A plasmid capable of receiving afragment of DNA and showing a controlled constant plasmid copy numberwhen host bacteria carrying the plasmid are cultivated at onetemperature, and showing, when the host bacteria carrying the plasmidare grown at a different temperature, an altered plasmid copy numbercontrol allowing an at least 20 times higher or totally uncontrolledcopy number.